Functionally graded biocompatible coating and coated implant

ABSTRACT

The present invention provides a biocompatible coating comprising calcium phosphate that is functionally graded across the thickness of the coating. The coating, which preferably includes hydroxyapatite, is particularly useful for coating implants, such as dental or orthopedic implants. The functionally graded coating is generally crystalline near the interface with the surface of the implant, with crystallinity and crystal diameter decreasing toward the outer layer of the coating. The invention further provides methods for preparing a coated implant comprising a functionally graded calcium phosphate coating thereon.

FIELD OF THE INVENTION

The present invention relates to biocompatible coatings. In particular,the invention is related to implants coated with a biocompatible calciumphosphate coating and methods of preparation of such coated implants.

BACKGROUND OF THE INVENTION

Various types of implants are commonly used in biomedical applications,particularly in the dental and orthopedic fields. Often, implants areassociated with use in areas of hard tissue (i.e., cartilage, bone,etc.), and the implants generally comprise hard, durable materials, suchas metals, particularly titanium.

Uncoated titanium implants are normally covered by a bioinert surface oftitanium dioxide. The presence of the bioinert surface structureprohibits biointegration of the implant by the surrounding tissue.Accordingly, the body responds to the foreign object by isolating theimplant with a flexible layer of fibrous tissue that can easily cause animplant to loosen. This is detrimental to the usefulness of the implant.For example, in the case of dental implants, loosening of the implantcan result in loss of the implanted tooth and can also lead toinfections around the loosened implant.

It is commonly known in the art to apply various coatings to orthopediccomponents and other medical devices for a variety of reasons, includingfacilitating implant fixation and bone in-growth. See, Handbook ofMaterials for Medical Devices, Davis, J. R. (Ed.), Chapter 9,“Coatings”, (2003). In particular, calcium phosphate phases are usefulas coatings for facilitating bone in-growth. One calcium phosphatephase, hydroxyapatite (HA) [Ca₁₀(PO₄)₆(OH)₂], is the primary mineralcontent of bone and calcified cartilage, representing 43% by weight ofbone. Because of the chemical and crystallographic similarities with theinorganic components of bone, applying a thin layer of HA, or othercalcium phosphate layer, to the surface of a metal implant, such as atitanium implant, can promote osseointegration and increase themechanical stability of the implant. In fact, many studies havedemonstrated that dental and orthopedic implants coated with plasmasprayed HA promote greater direct bone attachment and higher interfacialstrength compared to the uncoated titanium implants. Numerous problemswith the HA coatings, however, have also been cited, including variationin bond strength at the coating-metal interface, variation in structuraland chemical properties, and non-uniformity in coating density.

Hydroxyapatite coatings are generally comprised of varying percentagesof crystalline HA, tricalcium phosphate, and amorphous calciumphosphate. The ratio of HA to tricalcium phosphate has been reported tobe crucial for bone regeneration. It has also been reported that thedissolution rate of a HA coating is correlated to the biochemicalcalcium phosphate phase of the coating. It is known that coatings withmore crystalline HA are more resistant to dissolution. Conversely,coatings with increased concentrations of amorphous calcium phosphateand tricalcium phosphate are thought to predispose the HA coatings todissolution. Since it has been suggested that the dissolution of calciumphosphate from the surface of the implant in the body is responsible forthe bioactivity of the HA coating, knowledge of the crystalline contentof the surface coating is critical to implant success. Some studies haveindicated that bone responds differently to HA coatings of differentcrystallinity. These studies have indicated higher bone activity withwell characterized HA coatings of higher crystallinity, while otherstudies suggest that some amorphous phase in the coatings is desirableand promotes a more stable interface with the biological environment.Still further studies have identified various structural factors thatalso affect the biological response of bone to HA coatings, includingsurface texture, porosity, and the presence of trace elements.Accordingly, it is beneficial for the characteristics of the implantsurface to be precisely controlled during the implant process,particularly with respect to the crystalline content of the coatingsurface.

Traditional HA coatings are deposited by various techniques, such assputtering, electron beam deposition, laser deposition, and plasmaspraying. Because of its simplicity and versatility, plasma spraying isthe most widely used technique. Although plasma spraying is fast andcost effective, the coatings have several flaws that could lead toimplant failures. Plasma sprayed films exhibit a high porosity and onlyattach to the substrate surface through mechanical bonding (i.e., nointermolecular bonding). This leads to inconsistent bonding strengths.Further, regardless of the coating methodology, amorphous layers aregenerally formed on metal substrates, which have a high dissolution ratein aqueous solutions. Therefore, the layers are often subsequentlyheat-treated at approximately 600° C. to convert the amorphous phaseinto a crystalline phase. This heat treatment, however, causes cracks inthe layer due to the thermal expansion mismatch between the coated layerand the metal substrate. This leads to a severe reduction in bondstrength.

Plasma sprayed coatings are also relatively thick. Generally, coatingson commercially available plasma sprayed implants have a thickness ofbetween 79 μm and 111 μm. Such thick coatings can lead to low fractureresistance. This, along with reduced bond strength, can lead todelamination, and detached fragments have very adverse effects on theimplant, as well as the tissue surrounding it. For example, particulatedebris at the bone prostheses interface with HA coated implants has beenfound to cause a foreign body response that is destructive to thesurrounding tissues. As a result, improvement of the HA coatingproperties may reduce shedding and possibly prevent an aggressiveosteolytic response. Some studies have indicated that thin HA coatings(about 2 μm) have a significantly greater coating-metal interfacialstrength compared to commercially available thick (70 μm) plasma sprayedHA coatings (40 MPa versus 9 MPa, respectively).

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided abiocompatible coating comprising a calcium phosphate film having aplurality of layers. In one particular embodiment, the film comprises abottom layer, one or more intermediate layers, and a top layer. Thecalcium phosphate film is functionally graded in at least one ofcrystallinity and crystal size (particularly crystal diameter). In oneparticular embodiment, the calcium phosphate film is functionally gradedin crystallinity and crystal size such that degree of crystallinity andcrystal size both decrease from the bottom layer to the top layer.

In one embodiment of the invention, the calcium phosphate is selectedfrom the group consisting of hydroxyapatite, tricalcium phosphate, andmixtures thereof. Preferably, the film is functionally graded such thatthe bottom layer comprises predominately crystalline calcium phosphatewith crystals in a given crystal size range, the one or moreintermediate layers comprise crystalline calcium phosphate with crystalsof a smaller size than in the bottom layer, and the top layer comprisesa mixture of crystalline calcium phosphate (the crystals being generallysmaller than in the one or more intermediate layers) and amorphouscalcium phosphate. Most preferably, the top layer is predominatelyamorphous calcium phosphate.

According to another aspect of the invention, there is provided abiocompatible coated substrate comprising a substrate having a surfaceand a biocompatible coating on the surface of the substrate. In oneembodiment, the biocompatible coating comprises a calcium phosphate filmhaving an inner layer bonded to the surface of the substrate, one ormore intermediate layers, and an outer layer. Preferably, the film isfunctionally graded in at least one of crystallinity and crystaldiameter such that crystallinity and crystal diameter decrease from theinner layer to the outer layer.

In one particular embodiment, the invention provides a coated dentalimplant comprising a dentally implantable substrate having a surfacethat is at least partially coated with a calcium phosphate film havingan inner layer bonded to the surface of the substrate, one or moreintermediate layers, and an outer layer, wherein the film isfunctionally graded in crystallinity and crystal diameter such thatcrystallinity and crystal diameter both gradually decrease from theinner layer to the outer layer. According to another particularembodiment, the invention provides a coated orthopedic implantcomprising an orthopedically implantable substrate having a surface thatis at least partially coated with a calcium phosphate film having aninner layer bonded to the surface of the substrate, one or moreintermediate layers, and an outer layer, wherein the film isfunctionally graded in crystallinity and crystal diameter such thatcrystallinity and crystal diameter both gradually decrease from theinner layer to the outer layer.

According to another aspect of the invention, there is provided a methodfor preparing a biocompatible coated substrate. In one embodiment, themethod comprises providing a substrate having a surface, heating thesubstrate to a beginning deposition temperature, applying a calciumphosphate film to the surface of the substrate, and manipulating thedeposition temperature during the applying step. The method is effectivefor forming a coating on the substrate comprising a calcium phosphatefilm having an inner layer bonded to the surface of the substrate, oneor more intermediate layers, and an outer layer, wherein the film isfunctionally graded in at least one of crystallinity and crystal sizesuch that crystallinity and crystal size decrease from the inner layerto the outer layer.

In one particular embodiment, the film is functionally graded such thatthe degree of crystallinity decreases from the inner layer to the outerlayer. In still another embodiment, the film is functionally graded suchthat degree of crystallinity and the crystal size both decrease from theinner layer to the outer layer.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is more fully illustrated by the followingexamples, which are set forth to illustrate the present invention andare not to be construed as limiting.

FIG. 1 is one embodiment of a dual ion beam sputtering system useful fordepositing a coating according to the invention on a substrate;

FIG. 2 is a TEM image of a cross-section of a hydroxyapatite filmaccording to the invention coated on a silicon substrate;

FIG. 3 is a detailed view of the TEM image of FIG. 2 showing theinterfacial region between the HA coating and the silicon substrate;

FIG. 4 is a detailed view of the TEM image of FIG. 2 showing the nanocolumnar HA crystals underneath the amorphous HA;

FIG. 5 is a TEM image of the cross-section of a hydroxyapatite filmaccording to the invention marked at various areas from which SADpatterns were taken;

FIGS. 6 a-6 d are SAD patterns from areas indicated in FIG. 5 aslocations 1 to 4, respectively; and

FIG. 7 is a chart illustrating percentage cell adhesion versus time forcells incubated on a hydroxyapatite coating according to one embodimentof the invention.

DETAILED DESCRIPTION

The invention now will be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. These embodiments are provided so that thisdisclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art.

The present invention provides a functionally graded coating, coatedsubstrates coated with the functionally graded coating, and methods ofpreparing such coated substrates. The coating is characterized by agradual decrease in at least one of the crystallinity and the crystalgrain size (particularly the crystal diameter) across the thickness ofthe coating. The bottom (or inner) portion of the coating generallycomprises crystalline phase material of relatively large crystal size(compared to the remaining portions of the coating). The crystallinityand crystal size gradually decrease moving toward the top (or outer)portion of the coating leading to crystal grains of smaller and smallersize and a reduction in the extent of crystallinity, eventually becomingpredominately amorphous at the top (or outer) surface of the coating.

The coatings of the invention are described in terms of beingfunctionally graded across the thickness of the coating. Functionallygraded materials are understood to comprise materials wherein thecomposition, the microstructure, or both are locally varied so that acertain variation of the local material properties is achieved.Functionally graded coatings are particularly useful in that they can bestructurally engineered to allow for discrete or continual variations inthe molecular modeling of the coating. This allows for preparation ofcoatings with varying thermal, mechanical, and even bioactive propertiesacross the thickness of the coating. The present invention allows for aneven greater ability to engineer the coating on an atomic level tonanostructure the coating to predetermined specifications that maximizestrength and durability in one phase of the coating while maximizingbioavailability (e.g., osseointegration) in another phase of thecoating.

According to the present invention, functionally graded calciumphosphate coatings are provided. The coatings are functionally graded inthat at least one of degree of crystallinity and crystal grain sizechanges according to predetermined parameters across the thickness ofthe coating. Preferably, the coatings are functionally graded such thatat least one of degree of crystallinity and crystal grain size decreasesfrom the bottom layer of the coating to the top layer of the coating. Inone particular embodiment of the invention, the coating is functionallygraded in degree of crystallinity from the bottom layer to the top layersuch that the calcium phosphate is predominately crystalline in thebottom layer and crystallinity decreases moving toward the top layersuch that the calcium phosphate is predominately amorphous in the toplayer. Degree of crystallinity, as used herein, refers to the relativepercentage of the calcium phosphate material present that is in acrystalline phase versus that present in a non-crystalline phase (e.g.,an amorphous phase). A high degree of crystallinity would indicate thematerial present is predominately in a crystalline phase. A low degreeof crystallinity would indicate the material present predominately in anon-crystalline phase.

In another embodiment of the invention, the coating is functionallygraded such that both degree of crystallinity and crystal grain sizechanges according to predetermined parameters across the thickness ofthe coating. Preferably, degree of crystallinity and crystal grain bothdecrease from the bottom layer of the coating to the top layer of thecoating. In one particular embodiment, the coating comprises a bottomlayer, one or more intermediate layers, and a top layer. In thisembodiment, the bottom layer has a high degree of crystallinity andcrystal grain size of large size in relation to the remaining layers ofthe coating. In the one or more intermediate layers of this embodiment,the degree of crystallinity is less than or equal to the degree ofcrystallinity in the bottom layer, and the crystal grain size of thecrystalline material is less than the crystal grain size in the bottomlayer. In the top layer of this embodiment, the degree of crystallinityis less than the intermediate layer and the bottom layer. Preferably,the degree of crystallinity in the top layer is low, more preferably thematerial is predominately amorphous. Further, in this embodiment, thecrystal grain size of any crystals present in the top layer is smallerthan the crystal grain size in the intermediate layer or the bottomlayer.

Description in terms of crystal grain size can vary depending upon thetype of material present and the crystalline shape inherent to thematerial. In the present invention, calcium phosphate can be present invarious different embodiments. In one embodiment, the calcium phosphateis present as tricalcium phosphate (Ca₃(PO4)₂). In another embodiment,the calcium phosphate is present as hydroxyapatite. In yet anotherembodiment, both tricalcium phosphate and hydroxyapatite are present.Preferably, at least a portion of the calcium phosphate is in the formof hydroxyapatite. In one particular embodiment, the calcium phosphatecomprises predominantly hydroxyapatite. For purposes of simplicity, thecoatings of the invention may be described throughout in relation to anembodiment of the invention wherein the calcium phosphate ishydroxyapatite. Description of the coating in terms of comprisinghydroxyapatite, however, should not be interpreted as limiting thecoatings to that single embodiment. Rather, the coatings can compriseother calcium phosphate materials, as previously noted.

Both tricalcium phosphate and hydroxyapatite, when in crystalline thinfilm sputter deposited form, are generally columnar shaped crystals.Accordingly, crystal grain size for such columnar shaped crystals can bedescribed both in terms of crystal length and crystal diameter. As usedherein, crystal grain size is generally understood to relate to crystaldiameter. Accordingly, the terms crystal grain size and crystal diametercan be used interchangeably.

Preferably, the coatings of the invention are functionally graded suchthat crystallinity and crystal grain size decrease moving in the samedirection across the thickness of the coating. Accordingly, the coatingcan be characterized as comprising a bottom layer, a top layer, and oneor more intermediate layers. When the coating is applied to a substrate,the bottom layer is referred to as the inner layer as it is the layerinterfacing with the substrate surface. Further, when applied to asubstrate, the top coating layer is referred to as the outer layer.

The various layers comprising the coating can have well-definedboundaries. Alternately, the various layers can transition moregradually from one to another. Further, such transitioning can be sogradual such that the description in terms of “layers” is more of anabstract characterization of the coating used solely to describe thefunctionally graded state of the coating and is less of an actual visualdescription of the coating. Such transitioning can be strongly affectedby the method of preparation of the coating, which is described ingreater detail below. Further, it is possible for a single coating tohave portions where the transition is gradual and also have portionswhere the layer boundaries are well-defined.

In one aspect of the invention, the biocompatible coating comprising acalcium phosphate film having a bottom layer, one or more intermediatelayers, and a top layer. The film is functionally graded incrystallinity and crystal diameter such that crystallinity and crystaldiameter both decrease from the bottom layer to the top layer.

In one particular embodiment of the invention, the bottom layer of thecoating comprises predominately crystalline calcium phosphate.Preferably, the crystalline calcium phosphate in the bottom layercomprises crystals having a diameter of about 2 nm to about 50 nm. Morepreferably, the bottom layer comprises crystals having a diameter ofabout 5 nm to about 30 nm, most preferably about 5 nm to about 20 nm.Generally, the bottom layer can include crystals of diameter sizesvarying throughout the ranges noted above intermixed throughout thelayer. The crystals in the bottom layer preferably have a sizedistribution such that more crystals are of a size toward the upper endof the size range than toward the lower end of the size range.

The coating also can be functionally graded within an individual layer.For example in the bottom layer, the crystalline calcium phosphate nearthe bottom portion of the bottom layer can generally comprise crystalsof greater diameter than the crystals near the upper portion of thebottom layer. Additionally, an individual layer can be functionallygraded in terms of degree of crystallinity. In the bottom layer, whileit is preferred that the layer comprise predominately crystallinecalcium phosphate, the layer can, to some degree, also compriseamorphous calcium phosphate. Accordingly, the bottom layer can befunctionally graded within the layer such that the calcium phosphatenear the bottom portion of the bottom layer is at or about 100%crystalline, while the calcium phosphate near the top portion of thebottom layer includes a higher percentage of amorphous calciumphosphate. Such functional grading within a specific layer can also bepresent in the remaining layers of the coating of the invention,particularly in the one or more intermediate layers.

The thickness of the bottom layer can vary depending upon the intendeduse of the coating, the method of formation of the coating, and thesubstrate to which the coating may be applied. In one embodiment, thebottom layer of crystalline calcium phosphate has a thickness thatcomprises up to about 50% of the overall thickness of the coating. Inanother embodiment, the bottom layer has a thickness of about 50 nm toabout 1,000 nm.

The one or more intermediate layers particularly provide a transitionarea for the calcium phosphate coating. The one or more intermediatelayers can consist of a single layer that is, preferentially,functionally graded throughout. In other embodiments, two or moreintermediate layers can be present, each individual intermediate layerhaving a discrete composition (i.e., degree of crystallinity and rangeof crystal diameter), or each individual layer being functionally gradedthroughout. As used throughout, the intermediate layer may be describedin terms of a single layer. However, it is understood that the inventionencompasses the presence of one or more intermediate layers, eachpossibly taking on the description provided herein.

While the bottom layer can include crystalline and amorphous calciumphosphate, it is preferable for the bottom layer to be predominatelycrystalline calcium phosphate (i.e., close to 100%). In the intermediatelayer, however, there can be greater variations in the degree ofcrystallinity. Preferably, the portion of the intermediate layer nearthe bottom layer has a greater percentage of crystalline calciumphosphate and a lesser percentage of amorphous calcium phosphate.Further, preferably, the percentage of amorphous calcium phosphate inthe intermediate layer increases (and the percentage of crystallinecalcium phosphate decreases) through the intermediate layer moving awayfrom the bottom layer. However, in further embodiments, the intermediatelayer has a composition wherein the degree of crystallinity issubstantially uniform throughout the layer. In such embodiments, it ispreferable for the intermediate layer to have a greater percentage ofamorphous calcium phosphate than the bottom layer but have a lesserpercentage of amorphous calcium phosphate than the top layer.

Similarly, the crystal diameter of the crystalline calcium phosphatethroughout the intermediate layer is preferably generally smaller thanthe crystal diameter of the crystalline calcium phosphate in the bottomlayer. Again, the crystal diameter can be substantially uniformthroughout the intermediate layer, or the intermediate layer can befunctionally graded such that the crystal diameter of the crystallinecalcium phosphate decreases throughout the intermediate layer movingaway from the bottom layer. When the crystal diameter is substantiallyuniform, it is preferable for the crystal diameter generally to be lessthan the crystal diameter of the crystalline calcium phosphate in thebottom layer. When two or more intermediate layers are present, thecrystal diameter of the crystalline calcium phosphate can befunctionally graded across each of the intermediate layers, preferablysuch that the crystal diameter of the crystalline calcium phosphate isgenerally smaller in each succeeding intermediate layer moving away fromthe bottom layer.

Also similar to the bottom layer, the crystal diameter of thecrystalline calcium phosphate in the intermediate layer can vary acrossa range of diameter sizes. Preferably, a greater percentage of thecrystalline calcium phosphate in the intermediate layer has a crystaldiameter in the lower range of the crystal diameter size. In oneembodiment, the crystal diameter of the crystalline calcium phosphate inthe intermediate layer is about 2 nm to about 20 nm. Preferably, thecrystal diameter is about 2 nm to about 15 nm, more preferably about 2nm to about 10 nm. While there can be some overlap in crystal diameterbetween the bottom layer and the intermediate layer, the crystaldiameter of the calcium phosphate in the intermediate layer is generallysmaller than the crystal diameter of the calcium phosphate in the bottomlayer.

As with the bottom layer, the thickness of the intermediate layer canvary depending upon the intended use of the coating, the method offormation of the coating, and the substrate to which the coating may beapplied. Further, the thickness of the intermediate layer can varydepending upon the number of intermediate layers present, which canaffect how gradually, or how quickly, the coating is graded across thethickness of the coating. In one embodiment, the intermediate layer ofcrystalline calcium phosphate has a thickness that comprises up to about50% of the overall thickness of the coating. In another embodiment, theintermediate layer has a thickness of about 50 μm to about 1,000 nm.

As the coating is functionally graded across the thickness of thecoating, it can generally be characterized as transitioning from onestructural state near the bottom of the coating to another structuralstate near the top of the coating. As described above, the coating ispreferably predominately crystalline calcium phosphate with relativelylarge crystal diameter near the bottom of the coating. Accordingly, thecoating is preferably predominately amorphous calcium phosphate near thetop of the coating.

The top layer of the coating, as with the previous layers, can have acomposition that is substantially uniform throughout the layer inrelation to the degree of crystallinity and the crystal diameter of thecrystalline calcium phosphate present in the top layer. Alternately, thetop layer can be functionally graded across the thickness of the layer,the percent of amorphous calcium phosphate being lesser near the bottomportion of the top layer and being greater near the top portion of thetop layer. Preferably, the calcium phosphate in the top layer ispredominately amorphous calcium phosphate. In one embodiment, at leastabout 50% of the calcium phosphate in the top layer is amorphous calciumphosphate. Preferably, at least about 75% of the calcium phosphate inthe top layer is amorphous calcium phosphate, more preferably at leastabout 90% of the calcium phosphate in the top layer is amorphous calciumphosphate. In one embodiment, the top layer of the coating consistsessentially of amorphous phase calcium phosphate.

While is preferable for the calcium phosphate in the top layer to bepredominately amorphous calcium phosphate, it is possible for at least aportion of the calcium phosphate in the top layer to be crystallinecalcium phosphate. Accordingly, it is also preferred for the crystallinecalcium phosphate in the top layer of the coating to have a crystaldiameter generally that is less than the crystal diameter generallyfound in the intermediate layer or in the bottom layer. Again, whilethere can be an overlap in the ranges of crystal diameter sizes acrossthe various layers, preferentially, the crystal diameter in the toplayer is generally less than in the remaining layers. In one embodiment,the crystal diameter of the crystalline calcium phosphate in the toplayer is less than about 15 nm. Preferably, the crystal diameter of thecrystalline calcium phosphate in the top layer is less than about 10 nm,more preferably less than about 5 nm, and even more preferably, lessthan or equal to about 2 nm.

As before, the thickness of the top layer can vary depending upon theintended use of the coating, the method of formation of the coating, andthe substrate to which the coating may be applied. In one embodiment,the top layer of crystalline calcium phosphate has a thickness thatcomprises up to about 50% of the overall thickness of the coating. Inanother embodiment, the bottom layer has a thickness of about 50 nm toabout 1,000 nm.

In addition to calcium phosphate, it is possible for the coating of theinvention to include one or more additional components useful forincreasing the biocompatibility, including osseointegration, of thecoating. In one embodiment, the coating can comprise at least onefurther component commonly found in physiological bone that exhibits ahigh affinity between the ion of the component with calcium in serum.Examples of such further components include, but are not limited tozinc, magnesium, and fluoride. Still further, the coating can includeother components not commonly found in physiological bone so long as thecomponent exhibits a high affinity to calcium in serum. Such componentsare generally known to increase the stability and mechanical propertiesof the hydroxyapatite, as well as increasing the initial calciumabsorption of the HA coating from serum. Initial calcium absorption hasbeen shown to be critical for promoting new bone synthesis as it leadsto the binding of specific proteins that selectively enhance bone cellformation in and around the coating, enhancement bone cell attachment tothe coating, and facilitation of proper function of the coating in boneintegration. In one particular embodiment of the invention, the calciumphosphate coating further comprises an amount of yttrium.

Preferably, at least one further component is present only as arelatively small percentage of the calcium phosphate coating (i.e., adoping amount). In one embodiment, the calcium phosphate coatingcomprises up to about 10%, on a molar basis, of yttrium. Preferably, thecoating comprises up to about 8%, on a molar basis, of yttrium, morepreferably about 6%, on a molar basis.

The calcium phosphate coating of the invention is particularly useful asa coating for a substrate, such as an implant. Accordingly, the presentinvention further provides coated substrates comprising a substratehaving a surface and a biocompatible coating covering at least a portionof the surface of the substrate. The coating comprises a calciumphosphate film having an inner layer bonded to the surface of thesubstrate, one or more intermediate layers, and an outer layer. The filmis functionally graded in at least one of crystallinity and crystaldiameter such that crystallinity and crystal diameter changes from theinner layer of the coating to the outer layer of the coating.Preferably, at least one of crystallinity and crystal diameter decreasesfrom the inner layer of the coating to the outer layer of the coating.In one particular embodiment, both crystallinity and crystal diameterdecrease from the inner layer of the coating to the outer layer of thecoating.

The substrate according to the invention can include any item or devicewherein the presence of a biocompatible coating, particularly a coatingproviding increased osseointegration, would be advantageous. Preferably,the substrate is an item or device implantable in an area whereinteraction or integration with bony formations is desired or expected.In one embodiment of the invention, the substrate includes a prostheticimplant. According to another embodiment, the substrate includes adental implant. In yet another embodiment of the invention, thesubstrate includes an orthopedic implant. Items useful as substrates forcoating according to the invention include, but are not limited to thefollowing: dental screws, cylinders, blades, plates, and posts; partialor total joint replacements, including hip, knee, shoulder, and anklereplacements; orthopedic screws, pins, plates, bolts, nuts, rods, nails,and wires; and other similar dentally or orthopedically implantablesubstrates.

The substrate coated according to the invention can comprise anymaterial generally recognized by one of skill in the art as being usefulas an implantable item or device. In particular, the substrate cancomprise a material generally recognized as being useful as an implantin or around bony formations. In one embodiment of the invention, thesubstrate comprises one or more metallic material. Particularly, thesubstrate comprises a material selected from the group consisting oftitanium, titanium alloys, cobalt/chromium alloys, steel, and mixturesthereof. The invention, however, is not limited to these particularembodiments; rather, the substrate can include various additionalmaterials that may provide additional desirable properties orfunctionality to the substrate.

The coated substrate of the invention is particularly useful in thedental orthopedic fields. Accordingly, in one embodiment of theinvention, the substrate comprises a titanium dental implant. In anotherembodiment, the substrate comprises a titanium orthopedic implant.

Coates substrates according to the invention are also useful generallyin the area of bone reconstruction. For example, the coated substratecould be used in a partial or total joint replacement, in replacement ofan area of missing bone, and as a piece for treating bone fracture, suchas a screw or plate.

The coated substrate of the invention derives particular advantages fromthe functionally graded coating applied to the substrate. In thisembodiment of the invention, the coating can be characterized ascomprising an inner coating layer interfacing with surface of thesubstrate, one or more intermediate layers, and an outer layer overlyingthe one or more intermediate layers.

The inner layer of the functionally graded coating comprisespredominately crystalline calcium phosphate of relatively large crystaldiameter, particularly at the interface of the inner layer with thesurface of the substrate. Maximizing crystallinity at the interface withthe substrate increases the strength and the lifetime of the coating onthe substrate.

In one embodiment of the invention, the crystallinity and crystaldiameter of the calcium phosphate coating gradually decreases movingaway from the surface of the substrate and toward to the outer surfaceof the coating. The transition from higher crystallinity and greatercrystal diameter to lesser crystallinity and smaller crystal diameter isparticularly seen moving from the inner layer, across the intermediatelayer, and into the outer layer. Accordingly, the intermediate layercomprises a mixture of crystalline and amorphous calcium phosphate, aswell as crystals with a diameter spanning a range of sizes. The outerlayer of the coating is predominately amorphous calcium phosphate, andany crystalline calcium phosphate is of a relatively small crystaldiameter.

Maximizing the amorphous nature of the calcium phosphate in the outerlayer of the coating increases the biocompatibility of the coating,particularly by facilitating faster calcium absorption. The amorphouscalcium phosphate exhibits greater biodegradability than crystallinecalcium phosphate. Accordingly, the amorphous calcium phosphate in theouter layer of the coating facilitates the creation of channels andpores in the coating through which osseointegration can take place.Accordingly, the ability of surrounding bone to directly bond with thecoated substrate is increased by the presence of the coating,particularly due to the presence of the outer, amorphous layer. Completebiodegradability of the coating, however, does not take place due to thepresence of the inner, highly crystalline layer, which stabilizes thecoating on the substrate. In this manner, the functionally gradedcoating facilitates integration and stabilization of the implant at thedesired site of implantation. Furthermore, the coating limitscompetitive cell function at the implantation site.

The description of the calcium phosphate coating previously provided isalso applicable to the calcium phosphate film of the biocompatiblecoating used on the coated substrate of the invention. Therefore, it isunderstood that the calcium phosphate film on the coated substrates ofthe invention includes each of the embodiments previously described inrelation to the coated substrate.

According to another aspect of the invention, there is provided a methodfor preparing a biocompatible coated substrate. In one embodiment, themethod comprises providing a substrate having a surface, heating thesubstrate to a beginning deposition temperature, applying a calciumphosphate film to the surface of the substrate; and manipulating thedeposition temperature during the applying step. According to thisembodiment of the invention, there is formed a coating on the substratecomprising a calcium phosphate film having an inner layer bonded to thesurface of the substrate, one or more intermediate layers, and an outerlayer, wherein the film is functionally graded in at least one ofcrystallinity and crystal diameter such that crystallinity and crystaldiameter decrease from the inner layer to the outer layer. Preferably,crystallinity and crystal diameter both decrease from the inner layer tothe outer layer.

In one embodiment of the invention, the calcium phosphate coating isapplied using an Ion Beam Assisted Deposition (IBAD) system. Preferably,the IBAD system comprises dual ion beam sputtering using a primary beamand an assist beam. One embodiment of a dual ion beam sputtering systemis provided in FIG. 1. It is understood, however, that the presentinvention is not limited to a single type of sputtering system, butcould rather be practiced with any number of similar systems readilyunderstood by one of skill in the art. In the embodiment shown in FIG.1, the primary ion source is an 8 cm Kaufman-type ion source, used forsputtering the source material from the target, and the secondary ionsource is a 3 cm Kaufman-type source, used for ion bombardment.

Use of IBAD provides multiple advantages over plasma spraying. Whenusing IBAD, the calcium phosphate film bonds to the surface of thesubstrate on an atomic level, which leads to better and more consistentadhesion strength than available with plasma sprayed coating. Byapplying the calcium phosphate coating with an IBAD system, the calciumphosphate is deposited on the surface of the substrate molecule bymolecule. This allows for the formation of intermolecular bonds, in partbecause of the use of the assist beam, which directs the individualmolecules to the surface of the substrate. Accordingly, the inner layerof the calcium phosphate film is bonded to the surface of the substratethrough intermolecular bonding with the substrate molecules. Further,such intermolecular bonding can occur between the coating molecules andthe substrate molecules at or beneath the surface of the substrate. Themolecule by molecule deposition of the calcium phosphate coating furtherallows for precise control of thickness and other physicalcharacteristics. Accordingly, use of the IBAD system for applying thecalcium phosphate coating to the substrate allows for much thinnercoating than can be applied using plasma spray techniques. Thinnercoatings can provide a higher interfacial strength and better fractureresistance than thicker plasma spray coatings.

In one embodiment of the invention, the coating applied to the substratehas an overall thickness of about 100 nm to about 2,000 nm. Preferably,the coating has an overall thickness of about 200 nm to about 1,500 nm,more preferably about 300 nm to about 1,000 nm. Coating thickness canvary depending upon the length of time of deposition, which can becontrolled within close limits. Accordingly, coatings of preciselydefined thicknesses can be prepared according to the method of theinvention. Further, the thickness of the individual layers within thecoating can also be controlled within close limits by varying the lengthof time of deposition in coordination with the manipulation of thedeposition temperature, as described below.

In addition to the advantages described above in relation to improvingmechanical strength and bonding with the substrate, the method of theinvention is further advantageous in relation to the ability to preparecoated substrates with a functionally graded coating. Accordingly, themethod of the invention provides the ability to prepare coatedsubstrates wherein the coating has a higher percentage of amorphousphase calcium phosphate near the outer surface of the coating (toachieve better osseointegration an bone formation) and a higherpercentage of crystalline calcium phosphate near the interface of thecoating with the substrate (to achieve better mechanical and bondingstrength). The coated substrate prepared according to the method of theinvention has a nano-scale grain structure that closely mimics thestructure of bone itself, thereby facilitating the in-growth of bonewith the coated substrate, and generally improving the success of animplanted item or device.

Preparing such a desirable functionally graded coating on a substrate isachieved through manipulation of the deposition temperature during theapplication of the calcium phosphate coating on the substrate. Thephysical state of the coating deposited on the substrate is in part afunction of temperature. Accordingly, the physical state of the coatingcan be varied throughout the thickness of the coating by varying thetemperature at the time of deposition of the coating on the substrate.

The method according to the invention can vary depending upon the natureof the material being applied. Preferably, the substrate to be coated isinitially heated to a predetermined beginning deposition temperature.The beginning deposition temperature can be any temperature known tocorrespond to a temperature useful for depositing a material in aspecific phase or state. In one embodiment, the beginning depositiontemperature is a temperature known to correspond to a crystallinephase-forming temperature of the material for deposition. Accordingly,the material first deposited on the substrate will comprise a highpercentage of crystalline material, such as calcium phosphate. In onepreferred embodiment, the calcium phosphate applied to the substrate ishydroxyapatite. Accordingly, in one embodiment, the crystallinephase-forming temperature is in the range of about 500° C. to about 800°C. Preferably, the crystalline phase-forming temperature is in the rangeof about 650° C. to about 750° C.

The temperature manipulation portion of the method comprises loweringand, optionally, raising the temperature as desired throughout thedeposition process to affect the phase or state of the material beingdeposited on the substrate to prepare a coating that is functionallygraded according to desired specifications. In particular, the use oftemperature manipulation in combination with an IBAD system leads toformation of coated substrates wherein the coating can be preparedwithin closely defined specifications, including coating thickness,composition, and phase.

In one particular embodiment of the invention, the manipulation stepcomprises lowering the deposition temperature. In the coating of thepresent invention, it is desirable to have calcium phosphate in acrystalline phase near the substrate interface and calcium phosphate inan amorphous state near the outer surface of the coating. Withhydroxyapatite, for example, it is known that deposition at highertemperatures will lead to formation of a crystalline HA layer, whiledeposition at lower temperature will lead to formation of an amorphousHA layer. Accordingly, in one embodiment of the invention, thetemperature manipulating step comprises lowering the depositiontemperature to an amorphous phase-forming temperature. Such lowering canbe stepwise (i.e., immediate lowering to a predetermined temperature) orcan be gradual. In one embodiment of the invention, the amorphousphase-forming temperature is in the range of about 250° C. to about 500°C. As would be readily recognizable by one of skill in the art, theamorphous phase-forming temperature (as well as the crystallinephase-forming temperature) could vary depending upon the exact chemicalnature of the material for deposition. Accordingly, the presentinvention is not limited to the specific temperature ranges providedherein, but also foresees that other temperature ranges specific to thematerial for deposition are also encompassed by the invention.

In one embodiment of the coating of the invention, it is beneficial toheat the substrate to a beginning deposition temperature, beginapplication of the calcium phosphate coating (to facilitate formation ofcrystalline calcium phosphate), and then lower the depositiontemperature (to facilitate formation of amorphous calcium phosphate).The invention, however, also encompasses methods wherein the temperaturemanipulation comprises raising the temperature from the beginningdeposition temperature. The invention also encompasses methods whereinthe temperature manipulation comprises alternately raising and loweringthe temperature through a varying number of cycles to achieve afunctionally graded coating with a more complex grading. For example, ifdesirable, alternating layers of crystalline and amorphous HA could beapplied by alternately raising and lowering the deposition temperature.

The method of the invention can further comprise manipulation of one ormore of the ion beams used in the deposition process. In one particularembodiment of the invention, the IBAD system comprises a dual ion beamsputtering system comprising a primary beam and an assist beam, each setaccording to predetermined parameters. In one particular embodiment, theprimary beam and the assist beam are each set at a predeterminedvoltage. The predetermined settings for the primary beam and the assistbeam can vary depending upon the substrate to be coated, the coatingmaterial, and the exact desired properties of the coated substrate. Oneor more of the parameters of the primary beam and the assist beam canthen be manipulated to further affect the nature of the coating on thesubstrate.

In one embodiment of the invention, the method can further comprisemanipulating the assist beam, such as by lowering the voltage from thebeginning voltage to a lower voltage. It is useful to begin thedeposition process with the assist beam set at a higher voltage tofacilitate better bonding strength at the interface of the coating withthe substrate. After a new atomic layer has been deposited on thesubstrate, it is useful, according to one embodiment of the invention,to lower the voltage of the assist beam to avoid disruption of the newlyformed coating layers. Preferably, the assist beam voltage is loweredafter a specified period of time known to correspond to a time usefulfor deposition of a strongly bonded inner coating layer. In oneembodiment of the invention, the assist beam voltage is lowered from thebeginning voltage after a time of about 5 minutes to about 15 minutes.

The method of the invention, in addition to applying a calcium phosphatecoating to a substrate, can also encompass the application of furthermaterials. In one embodiment, the method further comprises applicationof one or more additional components useful for increasing the stabilityand mechanical properties of the calcium phosphate or for increasing theinitial calcium absorption of the coating from serum. In one embodiment,the additional component includes yttrium.

The addition of the further components to the calcium phosphate coatingof the invention can be by any method known in the art. When the coatingis prepared according to the method of the invention, it is beneficialto include the additional component through doping of the target used inthe IBAD system. The target generally comprises the material ultimatelydesired to be applied to the substrate. For example, in one embodimentof the invention, the target comprises an amount of hydroxyapatite. Thetarget can further comprise an additional material particularly usefulfor holding the material to be sputtered. In a particular embodiment,the target comprises an amount of pressed hydroxyapatite held on acopper plate. Other types of backing material, or plates, can also beused, such as steel. The present invention, however, is not limited tothese specific embodiments and can also comprise further materials thatwould be evident to one of skill in the art.

As previously noted, the additional components added to the calciumphosphate coating, while beneficial, are generally intended to beincluded only in a doping amount. Accordingly, it is beneficial for themethod of the invention to be particularly adaptable for allowing theinclusion of a source for the one or more additional components. In oneembodiment of the invention, the additional component added to thecalcium phosphate coating is yttrium, and the yttrium is introduced tothe coating by overlying strips of elemental yttrium on thehydroxyapatite target. For example, in one embodiment, two yttriumstrips are placed in a cross pattern over the target, the strips beingof a particular size such that the coating applied to the substrateexhibits the desired percentage of yttrium doping.

According to this embodiment of the invention, the percentage yttrium inthe coating applied to the substrate can be determined according to thefollowing formula:Y %=[(Y _(sa) /T _(sa))×(Y _(sy) /HA _(sy))]×100wherein Y_(sa) is the surface of the yttrium strips; T_(sa) is the totaltarget surface area; Y_(sy) is the yttrium sputter yield; and HA_(sy) isthe hydroxyapatite sputter yield. Further, Y_(sa) can be determinedaccording to the following equation:Y _(sa)=[(Y %×T _(sa) ×HA _(sy))/Y _(sy)]×100wherein each variable is as defined above. Similarly, when othercomponents are added to the calcium phosphate, the percentage of thecomponent included in the final coating and surface area of thecomponent placed on the target can be calculated according to the aboveequations. Therefore, the invention readily encompasses methods forincluding multiple different components in the coating of the invention.

The invention is particularly suited for preparing coated dentalimplants, coated orthopedic implants, and other types of coatedprosthetics. Generally, the invention can be used in the preparation ofcoated substrates wherein the substrate is any of the various dental andorthopedic items previously noted. In one embodiment of the invention,there is provided a coated dental implant comprising a dentallyimplantable substrate having a surface that is at least partially coatedwith a calcium phosphate film having an inner layer bonded to thesurface of the dentally implantable substrate, one or more intermediatelayers, and an outer layer, wherein the film is functionally graded incrystallinity and crystal diameter such that crystallinity and crystaldiameter both gradually decease from the inner layer to the outer layer.In a particular embodiment, the calcium phosphate film coating thedentally implantable substrate includes calcium phosphate selected fromthe group consisting of hydroxyapatite, tricalcium phosphate, andmixtures thereof. In still another embodiment, the calcium phosphatefilm further comprises one or more additional component, such asyttrium.

According to another embodiment of the invention, there is provided acoated orthopedic implant comprising an orthopedically implantablesubstrate having a surface that is at least partially coated with acalcium phosphate film having an inner layer bonded to the surface ofthe orthopedically implantable substrate, one or more intermediatelayers, and an outer layer, wherein the film is functionally graded incrystallinity and crystal diameter such that crystallinity and crystaldiameter both gradually decease from the inner layer to the outer layer.In a particular embodiment, the calcium phosphate film coating theorthopedically implantable substrate includes calcium phosphate selectedfrom the group consisting of hydroxyapatite, tricalcium phosphate, andmixtures thereof. In still another embodiment, the calcium phosphatefilm further comprises one or more additional component, such asyttrium.

A functionally graded coating deposited on a substrate according to themethod of the invention has been prepared and examined to study themicrostructure and physiological performance thereof. Variouscharacterization techniques, such as profilometry, transmission electronspectroscopy (TEM), scanning transmission electron microscopy (STEM),nano-indentation testing, and microscratch testing were used to analyzethe coated substrates. Such preparation and analysis of the inventivecoating are described more fully in the examples provided below.

Experimental

The present invention is more fully illustrated by the followingexamples, which are set forth to illustrate various embodiments of theinvention and are not to be construed as limiting thereof.

EXAMPLE 1 Preparation of Coated Substrate

A coated substrate was prepared by depositing a hydroxyapatite film on asilicon substrate in a dual ion beam sputtering system as shown inFIG. 1. The base pressure of the dual ion beam sputtering system was setat 9×10⁻⁷ Torr. A 15.24 cm diameter hydroxyapatite target recessed intoa stainless steel holder was used as the hydroxyapatite source.

For the duration of the deposition process, the primary ion source wasset at 1000V, and a gas flow of 3 sccm was provided to each ion sourcebringing the background pressure of the system to approximately 4×10⁴Torr. The assist beam was initially set to 1000V. After 10 minutes ofdeposition, the assist beam was set to 400V, and it remained at thatsetting for the remainder of the deposition.

For the first 2 hours of deposition, the substrate heater was set at700° C. After two hours of deposition, the substrate heater was reducedto 500° C. After an additional 2 hours, the heater was completely turnedoff. During the final 30 minutes of deposition, the substrate wasallowed to gradually cool, the substrate temperature ultimately fallingto 250° C. at the end of the deposition. Total deposition time was 4.5hours. A summary of the deposition parameters is provided in Table 1.TABLE 1 Assist Beam Primary Beam Substrate Step Time Voltage VoltageTemperature 1 1-10 minutes 1000 V 1000 V 700° C. 2 10-120 minutes  400 V1000 V 700° C. 3 120-240 minutes  400 V 1000 V 500° C. 4 240-270 minutes 400 V 1000 V 500° C.-250° C.

EXAMPLE 2 Structural Analysis of Hydroxyapatite Film on Coated Substrate

The cross-sectional structure of the HA film prepared in Example 1 isshown in FIG. 2. The figure provides a TEM cross-sectional image of thefunctionally graded HA film. The film has an overall thickness of 875nm, and on visual inspection, the film can be seen to include a numberof distinct layers. The silicon substrate is covered by a thin amorphouslayer of silicon dioxide (about 12 nm thick), followed by a coarsecrystalline HA layer (210 nm thick). On top of that is a finecrystalline HA layer (140 nm thick), followed by a layer of amorphous HAmixed with very fine nano-structure HA grains (513 nm thick).

As can be seen in FIG. 2, the degree of crystallinity decreases from thebottom layer (in contact with the substrate) to the top layer. Thebottom layer comprises predominately crystalline HA, while the top layercomprises predominately amorphous HA. Further, the crystal grain sizealso decreases from the bottom layer to the top layer. Large nanocrystals are present in the bottom layer, smaller nano crystals arepresent in the intermediate layer, and very little crystallization isseen in the top layer. The functional grading of the coating is furtherseen in FIGS. 3 and 4, which provide detailed views of the interfacialregion between the HA coating and the silicon substrate, and thetransition between the nano columnar HA crystals and the amorphouscrystals, respectively.

The selected area diffraction (SAD) patterns seen from the variouslayers further confirm the decreasing crystallinity of the inventivecoating moving toward the top layer of the film. FIG. 5 shows a TEMimage of the cross-section of the coated substrate marked at the variousareas from which SAD patterns were taken. Comparison of the TEM imagewith the SAD patterns of locations 1-4 further illustrates thedecreasing crystallinity of the film moving away from the substrate andtoward the top layer of the film.

The SAD pattern from location 1, shown in FIG. 6 a, is a mixture of HAfilm and silicon substrate. A well-defined spot pattern is visiblearising from the silicon substrate with a (011) zone axis. The ringsnumbered 1-4 in FIG. 6 a correspond to polycrystalline HA with (113),(213), (501), and (423) planes, respectively. FIG. 6 b reveals a similarring pattern indicating the presence of crystalline HA in location 2 ofFIG. 5. The SAD patterns from location 3 (FIG. 6 c) and location 4 (FIG.6 d) are composed of diffused rings, indicating an amorphous phase,possibly mixed with very fine nano-crystals. The distances of thediffused rings shown in location 3 and location 4 are similar with thed-spacings in location 1 and location 2, indicating the material foundin location 3 and location 4 is also HA, albeit in an amorphous phase.STEM analysis from various layers of the functionally graded HA filmshowed a Ca/P ratio very close to 1.66, which is the ratio for pure HA.

EXAMPLE 3 Preparation of Comparative Coated Substrate

For use as a comparative to the coating of the invention, a calciumphosphate film was sputtered on a cleaned glass surface using a CMS-18radiofrequency magnetron sputtering system. The target used in thesputtering process was a 101.6 mm diameter sintered HA target on acopper backing. The base pressure in the sputtering chamber was about6.5×10⁻⁶ Torr. Sputter-deposition was performed using a process pressureof about 1.0 to about 1.5 mbar and a sputtering power of 200 W for 7hours. A coating rate of 60 nm per hour was observed. After sputtering,the coated samples were subjected to post-deposition heat treatment at500° C. for 30 minutes using a Thermolyne 48000 furnace to prepare acrystalline phase coating. The post-deposition heat treatment resultedin a crystallinity of 62% (+/−2%), which was confirmed by X-raydiffraction.

EXAMPLE 4 Adhesion Characteristics Comparison

Adhesion characteristics were compared for the functionally graded HAfilm prepared in Example 1 and the sputter-deposited film of 62%crystallinity prepared as described in Example 3. Adhesion wasdetermined using a CSM Microscratch Instrument. Scratch tests wereperformed under a linearly increasing load that increased from 0.01 N to3 N. The scratch length was set to 3 mm, and the scratch speed was setto 1 mm/minute. A diamond tip (20 μm tip diameter, Rockwell C geometry)was used for the scratch testing.

Scratch testing was performed in two samples of the functionally gradedHA film of the invention, as prepared in Example 1. In the firstfunctionally graded HA film sample, the first critical load occurred at0.30 N. At higher loads, continuous film cracking was observed. Nosignificant spalling or external transverse cracking was observed,indicating good adhesion of the film to the substrate under the givenloading conditions. The scratch tip reached the substrate in the firstsample at a load of 2 N, and plastic deformation of the siliconsubstrate was observed at that load. In the second functionally gradedHA film sample, film penetration again did not occur until a load of 2 Nwas achieved.

In the sputter-deposited film of 62% crystallinity prepared according tothe method of Example 3, film penetration occurred at 0.42 N, which ismuch lower than in the functionally graded HA film of the invention(film penetration at 2 N). These test results illustrate the increasedability of the coated substrate of the invention to resist cracking.

EXAMPLE 5 Nanohardness and Young's Modulus

Nanohardness and Young's modulus values for the functionally graded HAfilm prepared in Example 1 and the sputter-deposited film of 62%crystallinity prepared as described in Example 3 were determined usingan MTS Nanoindenter II® instrument. The samples were indented with a DCMtip with a radius of curvature of 20 mm. Indentations were performedusing a trapezoidal loading curve. The nanohardness and Young's moduluswere measured as a function of indentation depth. The maximum load wasvaried between 150 mN and 600 mN. A constant loading rate of 30 mN/s wasapplied. The tip was calibrated following the partial unloading method,and was cleaned with isopropanol between indentations. The modulus andhardness were determined using the Oliver-Pharr model.

Table 2 provides a summary of the average hardness and average Young'smodulus values for the several films at a 100 nm maximum indentationdepth. As in Example 4, two samples of a hydroxyapatite coated substrateaccording to the invention are compared against the sputter-depositedfilm of 62% crystallinity described above in Example 3. As a furthercomparative, reported values for sintered HA are also provided [see,Kumar, R. R. and Wang, M., Materials Science and Engineering A338,230-236 (2000)]. TABLE 2 Average Hardness Average Young's Film Substrate(GPa) Modulus (GPa) Inventive Functionally Silicon 6.472 132.998 GradedHA (sample 1) Inventive Functionally Silicon 6.391 121.514 Graded HA(sample 2) Sputtered and Glass 5.101 95.892 Annealed HA Sintered HA NA6.19 125

As seen in Table 2, the functionally graded hydroxyapatite coating ofthe invention generally provides higher modulus and hardness values thanthe sputter-deposited films. Further, these values are also generallygreater than the reported values for sintered hydroxyapatite.

EXAMPLE 6 Cell Adhesion and Differentiation on Inventive HydroxyapatiteCoating

Initial cell adhesion and cell differentiation on the coating preparedaccording to Example 1 was evaluated using ATCC CRL 1486 human embryonicpalatal mesenchymal cell, and osteoblasts precursor cell line. The cellswere seeded on the HA coating in 6 well culture plates at a density of50,000 cells/sample in Dubecco Modified Eagle's Medium (DMEM) andincubated.

After 0.5, 1, 2, 3, and 4 hours of incubation, the non-adherent cellswere removed and counted using a Coulter Counter. The percentage celladhesion was calculated by the following equation:Number of initial cells suspended−Number of non-adherent cells×100Number of initial cells suspendedFIG. 7 provides a chart of percentage cell attachment on the inventiveHA coating versus time. As a comparative, the chart further provides thepercentage cell attachment on a silicone surface. On the inventive HAcoating, optimal adhered cell concentration was observed after 2 hoursincubation. On the silicone surface, optimal adhered cell concentrationwas observed after 0.5 hours incubation.

Cell layer protein synthesis and alkaline phosphatase specific activitywere also measured at 4 days after cell confluence. Protein synthesiswas performed using the Pierce BCA protein assay. After 4 daysincubation, the protein production by cells cultured on the inventive HAcoating was 0.0004 μg/μl (+/−0.0001). In comparison, protein productionby cells cultured on the silicone control surface was 0.0005 μg/μl(+/−0.0002).

Alkaline phosphatase (ALP) production was determined from p-nitrophenolstock standard. The ALP specific activity (nmol ALP/g protein) wascalculated by normalizing ALP production to the total protein produced.After 4 days incubation, the ALP specific activity on the inventive HAcoating was 2.00 nmol/μg (+/−0.69). In comparison, the ALP specificactivity on the silicone control surface was 0.13 nmol/μg (+/−0.21).

As seen from the above data, cell adhesion on the inventive HA coatingreached a plateau, and remained there, after 2 hours incubation,indicating good cell adhesion on the HA coating. Further, the cells onthe HA coating exhibited significantly less total protein productioncompared to the control, but also produced significantly greateralkaline phosphatase specific activity as compared to the control(p=0.00042). These results indicate that the inventive HA coatingenhances osteoblast differentiation.

Many modifications and other embodiments of the invention will come tomind to one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing descriptions andassociated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims. Although specific terms areemployed herein, they are used in a generic and descriptive sense onlyand not for purposes of limitation.

1. A biocompatible coating comprising a calcium phosphate film having abottom layer, one or more intermediate layers, and a top layer, whereinsaid film is functionally graded in crystallinity and crystal diametersuch that crystallinity and crystal diameter decrease from said bottomlayer to said top layer.
 2. The biocompatible coating of claim 1,wherein said calcium phosphate is selected from the group consisting ofhydroxyapatite, tricalcium phosphate, and mixtures thereof.
 3. Thebiocompatible coating of claim 1, wherein said bottom layer comprisescrystalline calcium phosphate having crystals with a diameter in therange of about 2 nm to about 50 nm.
 4. The biocompatible coating ofclaim 1, wherein said one or more intermediate layers comprisecrystalline calcium phosphate having crystals with a diameter in therange of about 2 nm to about 20 nm.
 5. The biocompatible coating ofclaim 1, wherein said one or more intermediate layers comprise a mixtureof crystalline calcium phosphate and amorphous calcium phosphate.
 6. Thebiocompatible coating of claim 1, wherein said top layer comprisescalcium phosphate with at least 50% being in an amorphous phase.
 7. Thebiocompatible coating of claim 6, wherein said top layer comprisescalcium phosphate with at least 75% being in an amorphous phase.
 8. Thebiocompatible coating of claim 1, wherein said top layer consistsessentially of amorphous calcium phosphate.
 9. The biocompatible coatingof claim 1, wherein said coating further comprises one or moreadditional component in addition to said calcium phosphate.
 10. A dentalimplant comprising: a dentally implantable substrate having a surface,and a biocompatible coating according to claim 1 bonded to the surfaceof the dentally implantable substrate.
 11. An orthopedic implantcomprising: an orthopedically implantable substrate having a surface,and a biocompatible coating according to claim 1 bonded to the surfaceof the orthopedically implantable substrate.
 12. A biocompatible coatedsubstrate comprising: a substrate having a surface; and a biocompatiblecoating comprising a calcium phosphate film having an inner layer bondedto said surface of said substrate, one or more intermediate layers, andan outer layer, wherein the film is functionally graded in crystallinityand crystal diameter such that crystallinity and crystal diameterdecrease from said inner layer to said outer layer.
 13. The coatedsubstrate of claim 12, wherein said substrate is a prosthetic implant.14. The coated substrate of claim 13, wherein said prosthetic implant isselected from the group consisting of a dental implant and an orthopedicimplant.
 15. The coated substrate of claim 12, wherein said substratecomprises one or more metallic material.
 16. The coated substrate ofclaim 15, wherein said substrate comprises a material selected from thegroup consisting of titanium, titanium alloys, cobalt/chromium alloys,steel, and mixtures thereof.
 17. The coated substrate of claim 12,wherein said calcium phosphate film has a thickness of about 100 nm toabout 2,000 nm.
 18. The coated substrate of claim 17, wherein saidcalcium phosphate film has a thickness of about 200 nm to about 1,500nm.
 19. The coated substrate of claim 17, wherein said calcium phosphatefilm has a thickness of about 300 nm to about 1,000 nm.
 20. The coatedsubstrate of claim 12, wherein said calcium phosphate is selected fromthe group consisting of hydroxyapatite, tricalcium phosphate, andmixtures thereof.
 21. The coated substrate of claim 12, wherein saidinner layer of said film comprises crystalline calcium phosphate havingcrystals with a diameter in the range of about 2 nm to about 50 nm. 22.The coated substrate of claim 12, wherein said one or more intermediatelayers of said film comprise crystalline calcium phosphate havingcrystals with a diameter in the range of about 2 nm to about 20 nm. 23.The coated substrate of claim 22, wherein said one or more intermediatelayers of said film comprise a mixture of crystalline calcium phosphateand amorphous calcium phosphate.
 24. The coated substrate of claim 12,wherein said outer layer of said film comprises calcium phosphate withat least 50% being in an amorphous phase.
 25. The coated substrate ofclaim 24, wherein said outer layer of said film comprises calciumphosphate with at least 75% being in an amorphous phase.
 26. The coatedsubstrate of claim 12, wherein said inner layer of said film is bondedto said surface of said substrate through intermolecular bonding withsubstrate molecules at or beneath the surface of the substrate.
 27. Thecoated substrate of claim 12, wherein said film further comprises one ormore additional component in addition to said calcium phosphate.
 28. Amethod for preparing a biocompatible coated substrate comprising:providing a substrate having a surface; heating the substrate to abeginning deposition temperature; applying a calcium phosphate film tothe surface of the substrate; and manipulating the depositiontemperature during said applying step, thereby forming a coating on thesubstrate comprising a calcium phosphate film having an inner layerbonded to the surface of the substrate, one or more intermediate layers,and an outer layer, wherein the film is functionally graded incrystallinity and crystal diameter such that crystallinity and crystaldiameter decrease from the inner layer to the outer layer.
 29. Themethod of claim 28, wherein said applying step comprises Ion BeamAssisted Deposition.
 30. The method of claim 29, wherein the Ion BeamAssisted Deposition comprises dual ion beam sputtering with a primarybeam at a predetermined voltage and an assist beam at a predeterminedvoltage.
 31. The method of claim 30, wherein said calcium phosphate issputtered from a calcium phosphate target by said primary beam.
 32. Themethod of claim 31, further comprising introducing at least oneadditional component to said calcium phosphate film, wherein saidintroducing step comprising overlying one or more strips of the at leastone additional component on the calcium phosphate target.
 33. The methodof claim 32, wherein said one or more strips are of a predeterminedsurface area for introducing a predetermined concentration of the atleast one additional component to the calcium phosphate film.
 34. Themethod of claim 28, wherein said manipulating step comprises loweringthe deposition temperature.
 35. The method of claim 28, wherein thebeginning deposition temperature is a crystalline phase-formingtemperature.
 36. The method of claim 35, wherein the crystallinephase-forming temperature is in the range of about 500° C. to about 800°C.
 37. The method of claim 34, wherein said temperature manipulatingstep comprises lowering the deposition temperature to an amorphousphase-forming temperature.
 38. The method of claim 37, wherein theamorphous phase-forming temperature is in the range of about 250° C. toabout 500° C.
 39. The method of claim 28, wherein the calcium phosphatefilm is applied to a thickness of about 100 nm to about 2,000 nm. 40.The method of claim 39, wherein the calcium phosphate film is applied toa thickness of about 200 nm to about 1,500 nm.
 41. The method of claim39, wherein the calcium phosphate film is applied to a thickness ofabout 300 nm to about 1,000 nm.
 42. The method of claim 30, furthercomprising manipulating the voltage of the assist beam.
 43. The methodof claim 42, wherein said step of manipulating the assist beam voltagecomprises lowering the voltage from a beginning voltage after aspecified time period.